The present invention relates to a method for detection of a basic peptide. More specifically, the present invention relates to a method for detection of basic peptide by mixing a sample that potentially includes a basic peptide and a reagent that includes a denatured albumin and detecting the resultant turbidness due to a complex formation between the basic peptide and the denatured albumin. The present invention also relates to a reagent for detection of a basic peptide to be used for the method.
The concentrations in blood of certain basic peptides are known to alter depending on pathological conditions and show differences than those in a healthy normal state. To a group of such basic peptides typically belong ghrelin, brain natriuretic peptide (BNP), adrenocorticotrophic hormone (ACTH), atrial natriuretic peptide (ANP), bradykinin and like, and these peptides are regarded as useful disease markers in the field of clinical examination. For example, it has been known that the concentration of a basic peptide, ghrelin, observed in plasma from patients suffering from severe heart failure and those suffering gastric cancer with severe cachexia is decreased. The blood concentration of BNP is an important clinical index of heart failure and BNP is used as a marker for examination.
The basic peptides have been conventionally measured by immunological procedures such as enzyme immunoassay and electrochemiluminescence immunoassay. In these immunological procedures, the basic peptides are measured with antibodies that specifically recognize and bind to them. However, the procedures are complicated for these methods because a labeling substance needs to be attached to a complex of the antibody and the basic peptide in order to detect the complex.
On the other hand, Dennis et al. (Dennis MS et al., J. Biol. Chem. vol. 277, 35035-35043 (2002)) and Lowenthal et al. (Lowenthal MS et al., Clin. Chem. vol. 51, 1933-1945 (2005)) have reported non-immunological methods for detecting basic peptides in which the step for binding a labeling substance can be omitted. However, the methods reported require an expensive and large-scale mass spectrometer or surface plasmon resonance analyzer. Thus, the methods have remained to be widespread.
Dennis et al. and Lowenthal et al. have studied binding between naturally occurring albumin (non-denatured albumin) and basic peptides in order to discover pathological indexes, investigate pharmacological action and develop drug delivery systems.
Albumin is a protein naturally contained in, for example, egg white, serum or milk. For example, serum albumin is known to have physiological functions including regulation of blood osmotic pressure, and to circulate in blood after its binding to blood metabolites such as fatty acids, hematin or bilirubin, compounds such as drugs, or special peptides.
For example, Baczynskyj et al. (Baczynskyj L. et al., Rapid Commun. Mass Spectrom. vol. 8, 280-286 (1994)) have showed a possibility where a known blood hormone, bradykinin, bind to BSA. In addition, Muramoto et al. (Muramoto K. et al., Biochemistry. vol. 20, 3380-3385 (1981)) have reported that a blood hormone peptide, ACTH, binds to BSA.